The effect of cations, g-strophanthin and oligomycin on the labeling from [32P]ATP of the (Na+ + K+)-activated enzyme system and the effect of cations and g-strophanthin on the labeling from [32P]ITP and 32Pi

Add time:08/19/2019    Source:sciencedirect.com

1.1. The particle which contained the (Na+ + K+)-activated enzyme system was labeled with 32P from [32P]ATP, [32P]ITP and 32Pi. The labeling from these three different sources was influenced by Mg2+, Na+, K+, and g-strophanthin.2.2. With [32P] ATP there were two kinds of labeling which differed in that one, the unstable, decreased when all [32P]ATP was hydrolyzed, whereas the other, the stable, did not. With Mg2+, the unstable labeling was low and the stable high. Na+ increased the initial rate as well as the amount of unstable labeling and decreased the stable labeling; this effect was most pronounced with a low concentration of ATP. With Mg2+ + Na+ + K+, the unstable labeling was very low, and the stable labeling was lower than with Mg2+ + Na+. When K+ or unlabeled ATP was added to a prelabeled particle, the unstable labeling rapidly decreased, whereas the stable labeling remained unaffected.3.3. The enzyme system was also labeled from [32P]ATP at 0°, and the effects of the cations at 0° and at 37° were similar. However, at 0°, the decrease in labeling due to K+ was not accompanied by an increase in hydrolysis.4.4. With [32P]ITP, the effect of the cations on the labeling was similar to that found with [32P]ATP, but the rate of labeling and the amount of 32P incorporated per mg protein was lower.5.5. The highest labeling from 32Pi was found with Mg2+, then followed by Mg2+ + Na+, and the lowest labeling was found with Mg2+ + Na+ + K+. This is the same pattern as for the stable labeling from [32P]ATP. From the experiments with 32Pi it could be estimated that 5–30% of the stable labeling in the experiments with [32P] ATP came from 32Pi released in the test solution when [32P]ATP was hydrolyzed.6.6. g-Strophanthin with Mg2+ and [32P]ATP or [32P]ITP in the medium affected neither the labeling nor the hydrolysis. With [32P]ATP and Mg2+ + Na+, g-strophantin decreased the unstable and increased the stable labeling. In concentrations of g-strophanthin giving a maximum effect, the labeling with Mg2+ + Na+ + g-strophanthin was identical with that of Mg2+ + Na+ + K+ + g-strophantin.7.7. With [32P]ATP and Mg2+ + Na+ at 0°, g-strophanthin decreased the labeling to the level found with Mg2+ + Na2+ + K+ and decreased the hydrolysis. The concentration of g-strophanthin necessary to decrease the labeling was lower than that which was necessary to give the same relative decrease in hydrolysis. With Mg+ + Na+ + K+, g-strophanthin had no effect on the labeling and caused only a very slight decrease in hydrolysis.8.8. With [32P]ITP, the effect of g-strophanthin was similar to that with [32P]ATP at 37°. With 32Pi and with Mg2+ + Na+ + K+, g-strophanthin increased the labeling to a level which was higher than with Mg2+ without g-strophanthin. This contrasted with the stable labeling from [32P]ATP, which, with Mg2+ + Na+ + K+ + g-strophanthin, was between the labeling with Mg2+ and Mg2+ + Na+.9.9. At 37°, oligomycin, like g-strophanthin, had no effect on the labeling or on the hydrolysis with Mg2+, but decreased the hydrolysis with Mg2+ + Na+ and with Mg2+ + Na+ + K+. In contrast to the effect of g-strophantin, it increased both the unstable and the stable labeling with Mg+ + Na+; it also increased both the unstable and the stable labeling with Mg2+ + Na+ + K+ to a level aproximating that obtained with Mg2+ + Na+ without oligomycin.10.10. The unstable labeling showed that there was a phosphorylation followed by a dephosphorylation of the enzyme particle. The similarity between the effects of the cations and of g-strophanthin on the phosphorylation and on the hydrolysis of ATP suggested that the (Na+ + K+)-activated enzyme system was responsible for the phosphorylation. From the experiments it was not possible to tell if the stable labeling was also catalyzed by this enzyme system or was due to another enzyme which used the same substrate but had a different rate constant.11.11. It is discussed whether the phosphorylation and dephosphorylation found with Mg2+ + N+ in the medium are part of the same hydrolysis pathway as when the system is activated by Na+ + K+, or whether they are due to a pathway of hydrolysis found under conditions where the system gives a Na+ − Na+ exchange.

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